Rescue of conformational dynamics in enzyme catalysis by directed evolution

Area of Science:

• Enzymology
• Protein Engineering
• Biophysics

Background:

• Rational design and directed evolution are key for enhancing enzyme catalytic efficiency.
• Understanding the role of protein dynamics in enzyme function is challenging due to macromolecular flexibility.
• Identifying specific conformational changes linked to function requires advanced techniques.

Purpose of the Study:

• To investigate how second-shell mutations impact the catalytic activity of the proline isomerase CypA.
• To elucidate the mechanism by which local perturbations influence allosteric networks and protein dynamics.
• To correlate evolutionary selection for increased catalysis with changes in conformational dynamics.

Main Methods:

• Directed evolution was employed to screen for improved catalytic activity in an impaired CypA mutant.
• Nuclear Magnetic Resonance (NMR) spectroscopy was used for kinetic analysis.
• Room-temperature X-ray crystallography was utilized for structural determination.

Main Results:

• Two second-shell mutations were identified that partially restored the catalytic activity of CypA.
• Local mutations were shown to propagate through an allosteric network, affecting conformational dynamics.
• An accelerated interconversion between catalytically essential conformational sub-states was observed and correlated with increased catalysis.
• High-resolution X-ray ensembles captured both functional conformational sub-states.

Conclusions:

• Subtle genetic modifications can fine-tune enzyme function by altering protein dynamics.
• Directed evolution can reveal evolutionary pathways for optimizing enzyme catalysis.
• The study provides insights into the relationship between protein structure, dynamics, and catalytic activity.